Position vector

Position vectors are specialized destination vectors that can be efficiently used to Gibson assemble large biological circuits. Each position vector contains a chromatin insulator sequence, a Gateway recombination cassette, and a polyadenylation sequence.

These DNA motifs are flanked by two different, 40 base pair long, unique nucleotide sequences (UNS). The UNSs are flanked in turn by two I-SceI restriction sites. This arrangement of DNA motifs allows for fast and reproducible assembly of large biological circuits.

Entry vectors with circuit parts can be assembled into the position vector using the gateway cloning technique. SceI digestion of the resulting plasmid will yield a fragment with the circuit parts and the afore mentioned DNA motifs, flanked by the two UNSs. These fragments can be used for Gibson assembly right away. The uniqueness of the UNSs ensure specific assembly of a large number of different fragments.

The following steps are necessary to assemble a large circuit from position vectors:

• Produce entry vectors of all circuit parts

• Gateway cloning of circuit parts into the postion vector

• SceI digestion

• Gibson assembly of the complementary UNSs